Aptamers, derived from the Latin aptus, meaning, ‘to fit’, are oligonucleotides that have a specific three dimensional shape and consequent biological activity. Aptamers are generally produced through a process named “systematic evolution of ligands by exponential enrichment” or “SELEX,” which is an iterative selection and amplification process. Nucleic acids that bind to a target are selected (non-binders are simply washed away) and then subjected to a round of amplification. As this process is reiterated, tightly binding aptamers are enriched in the population, and extremely tight and specific binding between the aptamer and the target can be achieved. The reader is referred to U.S. Pat. No. 5,270,163 and the very large family of related patents for detailed SELEX protocols.
The extraordinary capacity of aptamers to bind tightly to specific targets underlines their tremendous potential as molecular therapeutics. For example, aptamers can be used to selectively target cells (such as tumor cells or pathogens) for death.
For example, U.S. Pat. No. 6,566,343 discusses the potential for aptamers directed at cell surface components of bacteria, cancer cells and parasites to activate the complement system and bring about the lysis of target cells. The patent discloses the linkage of two aptamers—one directed against the target cell and a second one against a component of the complement system (thus recruiting the complement cascade to the target cell)—to achieve complement activation and targeted cell death.
There are two distinct disadvantages to this approach. First, the aptamer-aptamer conjugates are subject to degradation from serum nucleases and second, the aptamer-aptamer conjugates are subject to rapid clearance by the kidneys. Thus, although aptamers are a powerful targeting system, in vivo nucleic acid stability remains a problem.
A Canadian team of researchers (Dougan et al., 2000) demonstrated that 3′-biotinylation of DNA significantly increased its resistance to serum nuclease activity. This was presumably due to steric hindrance and suggests that any 3′ or 5′ capping or nucleic acid modification should improve nucleic acid stability in vivo.
However, our research surprisingly indicates that 5′-biotinylation is not very effective against serum degradation of DNA, nor is the incorporation of 2′-Fluoro-modified deoxynucleotide triphosphates (2′F-dNTPs). Thus, the stability issue is not as simply addressed as one might predict. Hence, improved methods of stabilizing nucleic acids for in vivo therapeutic use are still needed and the invention addresses this problem.
While many researchers have utilized addition of primary aliphatic amines and other functional groups to the 3′ ends of solid-phase synthetic DNA, Vaijayanthi et al. (Indian Journal of Biochem. And Biophysics. Vol. 40, p. 382, 2003) teach that “3′-terminal modifications are somewhat difficult to achieve, as these days, most of the syntheses are being carried out on solid supports and the 3′-hydroxyl function is inaccessible for the desired modification to be incorporated. Moreover, the 3′-hydroxyl group is not sufficiently nucleophilic for introducing modifications during post-synthesis work-up.” The presently revealed invention identifies a facile means for direct attachment to the 3′ ends of double-stranded PCR products, thereby eliminating the difficulty in attaching to the 3′ ends of DNA oligonucleotides produced by solid-phase synthesis.
In addition to the difficulties with attachment of functional groups to the 3′ ends of solid-phase synthetic single-stranded DNA oligonucleotides, the oligonucleotides are limited in length to approximately 100 bases. This problem stems from the maximal 99% coupling efficiency which causes yields to be quite low for longer oligonucleotides. Yields can be theoretically estimated as (0.99)n where n is the length of the oligonucleotide in bases. Hence, an oligonucleotide of 100 bases in length would yield (0.99)100 or 0.366 (about 37% yield). This is a severely limiting factor for the mass production of lengthy anti-sense and artificial gene DNA conjugates. Again, the natural solution to such a problem lies in PCR which because of its enzymatic nature (using Taq DNA polymerase) is capable of synthesizing DNA amplicons that are hundreds to thousands of bases in length.
A further problem with solid-phase DNA synthesis that makes it impractical at present for large-scale industrial or pharmaceutical use is cost. A gram of solid-phase synthesized DNA can cost several thousand dollars. To deal with the problem, Vaijayanthi et al., 2003 and Pons et al. 2001 teach the strategies of reusable DNA synthesis columns and multiple synthesis columns in parallel to enhance overall productivity.
A revolutionary approach to the issue of large-scale DNA synthesis cost has been recognized by Vandalia Research Corp. which published in Genetic Engineering and Biotechnology News (Dutton, 2009) that it uses scaled-up PCR (via its Triathlon system) for the cost-effective mass production of 1 gram or more of DNA per machine per day. It is the primary intention of the present invention to provide a facile and cost-effective means to directly couple peptides, proteins and other useful molecules to the 3′ adenine overhanging ends of PCR products made by Vandalia Research or other industrial entities to lower the cost of large-scale short and lengthy DNA-3′-conjugates for the future pharmaceutical industry.